In general, the means of producing compounds include chemical synthesis and enzymatic synthesis. In order to produce the compounds which can be materials for a variety of pharmaceutical products, it is essential to efficiently carry out regiospecific and stereospecific modifications of starting compounds. It is known that the enzymatic synthesis is superior in terms of these reactions.
In order to utilize an enzyme as a practical catalyst at an industrial level, however, the fundamental margin of the enzyme has to be considered. It comprises a short life span of the enzyme and the necessity of a coenzyme for catalytic events. Much attention has been paid so far to the way to elongate the life span of the enzyme and to retain the enzyme activity in the design for manufacturing processes with the enzyme. In addition, most enzymes used for the enzymatic synthesis require coenzymes in the catalytic events, and, for example, enzyme reactions for oxidation-reduction require a pyridine nucleotide such as NADH. These coenzymes are expensive in general, and therefore the addition of coenzyme has become an economically major issue when performing enzyme reactions at an industrial level.
As a solution to overcome of the margin of these enzyme reactions, a strategy using cells of a microorganism, particularly of E. coli, as a field for the enzyme reaction has been developed. That is, by means of transforming E. coli with an enzymatic gene, the target enzyme can be abundantly expressed within the cell. This means that the enzyme is continuously produced in the cell, and the enzyme activity can be retained as long as the cell is alive. Furthermore, a variety of intracellular reactions of metabolism enable the coenzymes required for the enzyme reactions to be regenerated.
An attempt has been made to produce the chemical substances which can be varieties of pharmaceutical materials by means of “microbial conversion” which comprises cultivating such a transformant of E. coli in a culture medium and bringing the culture contact with a substrate compound to obtain the modified compound. In particular, the oxidation reaction by microbial conversion of a hydrophobic or amphipathic compound using E. coli which has been transformed with a cytochrome P-450 gene has importance in pharmaceutical manufacturing.
A cytochrome P-450 enzyme which is encoded with a cytochrome P-450 gene (hereinafter also simply referred to as a P-450 enzyme) is the generic term for a group of the protoheme containing protein which is bound to carbon monoxide in its reduced form to give the soret band at around 450 nm. The P-450 enzyme is bound to tissues of most animals and plants, microsome of molds and yeasts and mitochondrial inner membrane of a part of animal tissues, and it exists in some kinds of bacteria and molds in its soluble form.
The P-450 enzymes have a variety of substrate specificity. There are enzymes exhibiting extraordinary wide substrate specificity which can utilize a large variety of organic compounds as the substrate, whereas some enzymes are found to have a rather strict substrate specificity which reacts only with comparatively limited kinds of organic compounds. Also some show excellent selectivity in stereo-specificity or regio-specificity to the reaction site. In addition, it is known that the P-450 enzymes are involved in, as specific functions, a wide variety of reactions such as xenobiotic hydroxylation, epoxidation, dealkylation and denitrogenation within the cells exhibiting the P-450 enzymes by catalyzing the monooxygenation.
In particular, a part of the P-450 enzymes originated from microorganisms have practically been utilized for industrial production of useful compounds. One of the typical examples is the P-450 enzyme of Streptomyces carbophilus, which hydroxylates the 6α-position of compactin, a substrate, to produce pravastatin as a product which is a therapeutic agent for hyperlipidemia (see Non Patent Literature 1). Furthermore, the method of producing active vitamin D3 by hydroxylating the 1α-position and the 25-position of vitamin D3 utilizing the P-450 enzyme of the ATCC33795 strain of Pseudonocardia autotrophica has been put to practical use. These P-450 enzymes originated from the microorganism can catalyze the monooxygenation only by conjugating with the electron transport system (ferredoxin and ferredoxin reductase) which donates electrons to the enzymes.
Such microbial conversion of compounds using cytochrome P-450 enzyme originated from microorganisms has been performed by using a culture solution or bacterial body of the microorganism which was expressing the enzyme. Another culture solution has also been used which is given by introducing a gene encoding the P-450 enzyme originated from microorganisms into Streptomyces lividans suitable as a host, and caused to express its enzyme activity. However, the microbial conversion of a substrate compound by an actinomycete having such a gene requires considerable time for culturing and converting the substrate compound into the objective product because of the unique nature of actinomycetes. In addition, depending upon the enzyme, investigation of the expression inducing conditions is required for effectively increasing the expression level of the enzyme. Furthermore, some actinomycetes used for the conversion have a reaction system which metabolizes or degrades the substrate compound or the objective product, and this contributes to generation of byproducts and decrease in the substrate compound and the objective product to lower the productivity of the objective product.
For these reasons, it has been desired to establish a system which can functionally express the cytochrome P-450 gene originated from microorganisms and which uses as the host an E. coli requiring relatively short period of time for culture and also being considered to have less reaction systems to metabolize or degrade the substance compound and the objective product. As such a system, a system has been proposed that co-expresses the camAB gene encoding the electron transport system of P450cam and causes to functionally express the cytochrome P-450 gene of a wide variety of actinomycetes (see Patent Document 1). However, the activity of microbial conversion by this system was quite low and inadequate to be utilized in industrial production. Thus, in order to actually perform the industrial production by microbial conversion using E. coli which expresses the gene encoding the cytochome P-450 enzyme originated from microorganisms, further improvement of the activity has been required.
As the technique for it, the method of actively introducing the substance into the cell has been examined. For example, the E. coli caused to express the lat gene encoding L-lysine 6-aminotransferase originated from the IFO3084 strain of Flavobacterium lutescens is capable of converting L-lysine into L-pipecolic acid (see Non Patent Literature 2), and it is known that, in the microbial conversion using the transformant, the microbial conversion activity is improved by amplifying the lysP gene which encodes the L-lysine specific permease in order to actively introduce the substrate L-lysine into the cell (see Non Patent Literature 3).
Thus, in microbial conversion, it has been suggested that the membrane transport of substrate molecules into the cells is the important affair, but no mechanism exists in E. coli which actively perform the membrane transport into the cells the hydrophobic or amphipathic compounds which are frequently used as substrate compounds in microbial conversion for drug manufacturing. In addition, it has been reported that the intracellular abundance of the given compound (mammalian steroidal hormone) increases in the strain of E. coli subjected to disruption of drug efflux protein (see Non Patent Literature 4). This suggests the increase of the intracellular abundance on the ground that the compound passively penetrating into the cell becomes difficult to be eliminated from the body.
On the other hand, a patent application has been published of which the gist is that the E. coli subjected to transformation with at least one gene of acrA, acrB or tolC gene which encodes the drug efflux protein is provided with resistance to organic solvents, and the microbial conversion in the bilayer system using this E. coli can be performed with high efficiency (Patent Literature 2). These matters show it difficult to presume the effect of the disruption of multidrug resistant protein of the host microorganism on the microbial conversion.    [Patent Literature 1] Brochure of International Publication No. 2003/087381, its family in English US 2006234337 A1    [Patent Literature 2] Japan Patent Application Laid-open disclosure Kokai number Heisei 11-221080 (221080/1999A1)[Non Patent Literature]    [Non Patent Literature 1] Cloning, characterization and expression of the gene encoding cytochrome P-450sca-2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor. Gene. 1995 September 22;163(1):81-85.    [Non Patent Literature 2] Microbial conversion of L-lysine to L-pipecolic acid catalyzed by L-lysine 6-aminotransferase and pyrroline-5-carboxylate reductase. Biosci Biotechnol Biochem. 2002 March; 66(3):622-627.    [Non Patent Literature 3] Increase in the rate of L-pipecolic acid production using lat-expressing Escherichia coli by lysP and yeiE amplification. Biosci Biotechnol Biochem. 2002 September; 66(9):1981-1984.    [Non Patent Literature 4] Mammalian steroid hormones are substrates for the major RND- and MFS-type tripartite multidrug efflux pumps of Escherichia coli. J Bacteriol. 2006 February; 188(3):1191-1195.    [Non Patent Literature 5] Entry into and release of solvents by Escherichia coli in an organic-aqueous two-liquid-phase system and substrate specificity of the AcrAB-TolC solvent-extruding pump. J Bacteriol. 2000 September; 182(17):4803-10.    [Non Patent Literature 6] Production of alpha, omega-alkanediols using Escherichia coli expressing a cytochrome P450 from Acinetobacter sp. OC4. Biosci Biotechnol Biochem. 2006 June;70(6):1379-1385.
All the descriptions of the abovementioned Patent Literatures 1 and 2, and Non Patent Literatures 1 to 6 are expressly incorporated herein by reference in their entirety.
The object of the present invention is to provide a means for improving the low conversion efficiency shown in the present microbial conversion which uses the transformant inserted with a gene originated from xenogeneic organisms.